Deoxyribonucleic acid (DNA) copy number variation (CNV) is associated with certain genetic disorders, chromosomal rearrangements, and cancers.
The standard method for detection of CNV used routinely in the clinical lab is fluorescent in situ hybridization (FISH) [13,14]. The resolution of FISH is about 100 kilobases (kb), but CNVs involving shorter segments are difficult to detect with this method. In the last decade after completion of the human genome sequence [15], several molecular detection techniques capable of resolving shorter CNVs have revealed a remarkable degree of structural variation present among normal individuals.
The most popular techniques developed in the last decade are Comparative Genomic Hybridization (CGH) arrays, Single Nucleotide Polymorphism (SNP) arrays, real-time quantitative PCR (qPCR) and Multiplex Ligation-dependent Probe Amplification (MLPA) and massively parallel sequencing. Array-based techniques (CGH array and SNP array) are efficient for global and high resolution scans of structural features of human genome-wide variation [16-19]. The resolution of high density targeted arrays has approached to a few base pairs. In recent years, next-generation sequencing has been used for CNV detection [20,21]. The above methods are time consuming and require costly equipment and reagents.
Real-time qPCR can be used to calculate CNVs from the change in threshold crossing cycle number (ΔΔCq) [22,23]. This approach is rapid and needs no expensive instruments. Commonly observed ratios of copy number variations in the human genome can be 10:1 or greater, or as small as 4:3. The 3:2 ratios involved in trisomy (e.g., Down syndrome) are especially common. In theory, qPCR can be used to detect 2:1 CNVs and even trisomies, but considerable care is required in practice for reliable results, and it is very difficult to distinguish the smaller ratios by qPCR. qPCR is generally used for gene expression, however, for copy number determination, qPCR has been less used in the clinic.
MLPA has been widely used to detect CNVs associated with genetic disease in the clinical laboratory because it can detect large deletions and duplications in genes (typically deletions or duplications of exons) [24,25]. However, MLPA is time consuming and requires long customized oligonucleotide probes. All of these approaches, except real-time PCR, require at least one day to complete.
A need exists for rapid, alternative methods of determining CNVs and determining gene expression.